Nox2 is one of the seven members of NOX/DUOX family of NADPH oxidases with the biological function of generating Reactive Oxygen Species (ROS). Nox2-derived ROS have been involved in the pathogenesis of several disease conditions such as acute and chronic inflammation, cardiovascular diseases and aging. However, cellular ROS can be also produced by other members of Nox family and by other cellular enzymes such as xanthine oxidase, cytochrome P-450, mitochondrial oxydases etc. To date, the dissection of the contribution of Nox2-derived ROS to oxidants generated by other sources has been complicated by the lack of good specific Nox2 inhibitors. The purpose of this grant application is to identify new small-molecule inhibitors for Nox2 by screening chemical libraries in collaboration with TSRI Molecular Library Screening Center headed by Dr. Hugh Rosen. To this aim, we have developed a robust cell-based chemoluminescence (CL) assay in 96-well format for the detection of Nox2-derived upon phorbol-myristate acetate (PMA) stimulation. This assay will use COS-7phox as a cell line. Such cells have been genetically modified to stably express all the components required for Nox2 enzyme activity, and they represent a widely- accepted whole cell system capable of high level of PMA-induced ROS generation. We plan to implement this cell-based CL assay for High-Throughput Screening (HTS) in 384-well plate at the nearby MSLCN center. Furthermore, we propose to validate the primary hits with several follow-up assays, which we have successfully developed in 384-well plate. These secondary screens will i) eliminate non-specific or toxic hits ii) validate potential hits for their ability to block Nox2-dependent ROS generation in an independent cell line (Nox2-HEK293) iii) verify selectivity of primary hits to block the activity of other ROS-producing enzymes (i.e. xanthine oxidase) iv) assess the specificity of potential hits for Nox2 activity vs. activities of other members of Nox family. The compounds that emerge as validated Nox2-selective hits will be powerful investigative tools to study the involvement of Nox2-derived ROS in inflammation, cardiovascular diseases and aging. Consistent with this, we plan to use such newly-generated Nox2 inhibitors in combination with ongoing biochemical and functional analyses. Additionally, these inhibitors have the potential to provide novel therapeutic approaches to inhibit hypertension, aortic aneurysm, myocardial infarction, pulmonary fibrosis, arthritis, Alzheimer's disease, stroke, cancer, and inflammation. PUBLIC HEALTH RELEVANCE: It is known that NADPH oxidases (Nox2) play important roles in health and disease, yet much more remains to be learned about Nox2 function in biology. We will use a cell-based screen to identify Nox2 inhibitors, followed by secondary screens to verify selectivity, efficacy, and mechanism. Such inhibitors will be useful to investigate Nox2 biology, and as potential therapeutic agents in inflammatory and neurodegenerative diseases.